Novel Assay for Inositol Phosphorylceramide Synthase Activity

ABSTRACT

Disclosed is a simple and reproducible method for assaying inositol phosphorylceramide synthase activity that employs a fluorescence resonance energy transfer pair for measuring enzyme activity. The invention also includes a novel method for identifying IPC synthase inhibitors.

PRIORITY CLAIM

This application is a continuation application and claims priority underthe benefit of 35 U.S.C. §120 to U.S. Ser. No. 11/805,010 filed on May22, 2007 which claims priority to U.S. Ser. No. 60/802,479 filed on May22, 2006.

FIELD OF THE INVENTION

The invention relates to the field of antifungal agents, to methods forthe identification and characterization of compounds with the potentialto be developed into antifungal drugs. More specifically, the inventionrelates to methods for screening for inhibitors of inositolphosphorylceramide synthase (IPC synthase).

BACKGROUND OF THE INVENTION

The incidence of life-threatening fungal infections is increasing at analarming rate. With the exception of Staphylococci infections, thefungus C. albicans represents the fastest growing area of concern inhospital acquired infections. About 90% of nosocomial fungal infectionsare caused by species of Candida with the remaining 10% beingattributable to Aspergillus, Cryptococcus, and Pneumocystis. Whileantifungal compounds with some efficacy have been developed, progress indiscovery of new antifungal compounds has been slow and lags far behindthe rapid growth in the incidence of systemic fungal infections. (Seee.g. WO 2004/050685 PCT/US2003/038595 and references therein; Abi-Saidet al., 1996; Rees et al., 1998.) This is because of the innatedifficulties of finding selective inhibitors for one large group ofeukaryotes (fungi) invading another (humans). As a consequence, thecurrent antifungal drug market is dominated by only two classes ofdrugs, polyenes and azoles, both of which have significant limitations,in terms of efficacy, toxicity, problems with drug-drug interactions andthe generation of resistant organisms (e.g. Rex et al., 1995; Hay,2003). Hence, there is an urgent need for new antifungal compounds withnovel modes of action.

Inositolphosphoryl ceramides are sphingolipids found in a number offungi including but not limited to all major human pathogens (Lester andDickson, 1993; Vincent and Klig, 1995; Dickson and Lester, 1999).Organisms such as C. albicans, A. fumigatus, C. neoformans and H.capsulatum all contain inositolphosphoryl ceramides, as does S.cerevisiae, S. pombe, and N. crassa. The inositolphosphoryl ceramidebiosynthesis pathway in fungi involves at least eight separatereactions, each catalyzed be a specific enzyme. The first five steps inthe pathway, starting with the assembly of 3-ketohydrosphingosine, bythe enzyme serine palmitoyltransferase, and ending with thehydroxylation of ceramide, by ceramide hydroxylase, are quite similar tothe corresponding steps in the mammalian sphingolipid biosynthesispathway (Nagiec et al., 1997; Dickson and Lester, 1999). Consistent withthis, it has been found that inhibitors targeting these reactions haveclose to equal efficacy towards fungi and mammalian cells and,consequently, such compounds have little potential for development intoantifungal drugs. However, the sixth reaction step is unique to fungiand plants. In this step the enzyme IPC synthase catalyzes the transferof inositol phosphate from phosphatidyl inositol, to ceramide, to forminositol phosphorylceramide (FIG. 1). Genetic and mutational studieshave demonstrated that this reaction is essential in fungi (Nagiec etal., 1997). It has also been shown, in several organisms, thatinhibition of this reaction step is cidal (e.g Takesako et al., 1993;Endo et al., 1997). By contrast, it has also been demonstrated that thetwo to three downstream (from IPC synthase) reaction steps, in thefungal sphingolipid biosynthesis pathway, are not essential (Dickson andLester, 1999).

The uniqueness of the fungal IPC synthase (IPCS) catalyzed reaction,coupled with the fact that the enzyme is essential in fungi, make IPCsynthase an attractive target for antifungal drugs. Further supportingthis notion is the recent identification of several very potent naturalantifungal compounds that all target IPC synthase (e.g., Mandala 1997,Mandala 1998, Thong 1999, Kurome 2000). Some of these compounds havedemonstrated therapeutic activity in animal models, even when deliveredorally (Takesako 1993).

In view of the foregoing, inositol phosphorylceramide (IPC) synthase isan important enzyme in fungi and compounds capable of specificallyinhibiting this enzyme would have considerable potential to be developedinto antifungal drugs and hence meet an immediate, unmet medical need.

A significant impediment to the identification of novel IPC synthaseinhibitors with drug candidate potential, is the lack of an assaysuitable for robotized screening of large compound libraries(high-throughput screening). Currently used assays are complicated,labor intense and generate poorly reproducible data (e.g. Mandala etal., 1997; Mandala et al., 1998; Ko et al., 1995; Fischl et al., 1999;Aced et. al., 2004). As such they are completely unsuitable forhigh-throughput screening efforts.

SUMMARY OF THE INVENTION

This invention provides a method for assaying inositol phosphoceramidesynthase (IPC synthase) activity that is easy to perform, reproducibleand fully compatible with robotized high-throughput screeningprocedures.

The invention further comprises a novel methodology for screening, andother efforts aimed at identifying IPC synthase inhibitors with thepotential of becoming antifungal drugs.

In one aspect, the invention includes a method for measuring inositolphosphorylceramide (IPC) synthase activity, which includes providing asample containing inositol phosphorylceramide synthase, adding afluorescent labeled IPC synthase substrate, adding a phosphate bindingcompound conjugated to a fluorophor, wherein the phosphate bindingcompound conjugated to a fluorophor forms a FRET pair with thefluorescent labeled IPC synthase substrate, and measuring fluorescence.

In a second aspect, the invention provides a method for identifyinginositol phosphorylceramide synthase inhibitors, which includesproviding a sample containing inositol phosphorylceramide synthase,adding a test compound to the sample, adding a fluorescent labeled IPCsynthase substrate, adding a phosphate binding compound conjugated to afluorophor, wherein the phosphate binding compound conjugated to afluororphor forms a fluorescence resonance energy transfer (FRET) pairwith the fluorescent labeled IPC synthase substrate; and measuringfluorescence.

In a third aspect, the invention provides a kit that includes afluorescent labeled IPC synthase substrate and a phosphate bindingcompound conjugated to a fluorophor, wherein the phosphate bindingcompound conjugated to a fluororphor forms a FRET pair with thefluorescent labeled IPC synthase substrate. In one embodiment the kitfurther includes a sample containing inositol phosphorylceramidesynthase. In another embodiment the kit further comprises phosphatidylinositol, and in yet another embodiment, the kit further comprises aknown modulator of IPC synthase.

Specific embodiments of the foregoing aspects of the invention mayinclude one or more of the following. The fluorescent labeled IPCsynthase substrate is a fluorescent labeled ceramide. The fluorescentlabeled ceramide is selected from the group consisting ofC5-NBD-ceramide(5-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yeamino)hexanoyl)sphingosine),C6-NBD-ceramide(6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yeamino)hexanoyl)sphingosine),C12-NBD-ceramide(12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine),BODIPY-FL-C5-ceramide(N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine),BODIPY-FL-C6-ceramide and BODIPY-TR-C6-ceramide(N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine);all of which are available from Invitrogen, (Molecular Probes). Thefluorescent labeled ceramide is C6-NBD-ceramide. The phosphate bindingcompound includes a peptide that has at least one positively chargedamino acid. The phosphate binding compound includes a peptide that hasone or more of arginine, histidine and lysine. The phosphate bindingcompound includes a hexapeptide. The fluorophor conjugated to thephosphate binding compound is selected from the group consisting ofAlexa Fluor 350, anilinonapthalene, dansyl, dapoxyl, dibromobimane,diethylaminocoumarin, dimethylaminocoumarin, dimethylaminonapthalene,monobromobimane, monochlorobimane, napthanlene, pyrene, stilbene andD-346. The fluorescent labeled substrate is C6-NBD-ceramide and thefluorophor conjugated to the phosphate binding compound is D-346. Thesample containing inositol phosphorylceramide synthase is derived fromfungal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the reaction catalyzed by IPCsynthase.

FIG. 2 shows the chemical structure of C₆-NBD-ceramide.

FIG. 3 illustrates an example of the detection and quantification ofreagents functional in the IPC synthase FRET assay. The reagentcomprises the fluorophor D-346 conjugated to a peptide composed of sixarginine residues [in brackets].

FIG. 4 is a graph of absorption and emission spectra of examples offluorophors (FRET pairs) that can be used in IPC synthase FRET assay.The graph includes absorption and emission spectra of NBD, emissionspectrum of D-346, and examples of excitation and emission readwavelengths for the D-346/NBD FRET pair.

DEFINITIONS

A Fluorescence Resonance Energy Transfer (FRET) pair consists of twofluorophors wherein the absorption (or excitation) spectrum of onefluorophor overlaps the fluorescence emission spectrum of the secondfluorophor.

As used herein, an inositol phosphorylceramide (IPC) synthase substrateis a compound to which IPC synthase binds and transfers a phosphatecontaining group.

As used herein, a “test compound” refers to a substance such as achemical compound (naturally occurring or synthesized) such as abiological macromolecule, such as a nucleic acid (e.g., DNA, RNAantisense RNA, siRNA, and ribozymes), peptide, polypeptide,peptidomimetic, protein, non-peptide, antibody or fragment thereof,lipid, carbohydrate, small molecule, organic molecule, other drug or anextract made from biological materials such as bacteria, plants, fungior animal cells or tissues, or an inorganic element or molecules.

As used herein, a “sample” includes any material that contains theenzyme IPC synthase. For example, a sample can be an extract ormicrosomal membranes prepared from any fungal cells, including, withoutlimitation, Candida, Aspergillus, Cryptococcus, Saccharomyces,Neurospora, Histoplasmosis, Neurospora and Pneumocystis. A sample canalso be an extract or microsomal fraction from Trypanosoma cruzi orother protozoans or life forms that have IPC synthase activity(Figueiredo, et al. 2005). The sample can also be prepared from plantsor plant cells. A sample can also be purified IPC synthase or partiallypurified IPC synthase.

ABBREVIATIONS

NBD, 4-nitrobenzo-2-oxa-1,3-diazole

BODIPY,8-methyl-4,4-difluoro-1,3,5,7-tetramethyl14-bora-3a,4a-diasa-3-indacene

FRET, Fluorescence Resonance Energy Transfer

IPC, Inositol phosphorylceramide

DETAILED DESCRIPTION OF THE INVENTION

Current assays for IPC synthase activity involve incubatingphosphatidylinsitol andC₆—N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-ceramide (C₆-NBD-ceramide;FIG. 2), or radioactive ceramide, with an enzyme preparation, for acertain period of time. The reaction product, C₆-NBD-inositolphosphorylceramide (IPC), is subsequently separated from unreactedC₆-NBD-ceramide by adsorbing onto 100 μl (sedimented gel volume) AG4-X4resin, formate form, in a 96-well filter plate, using a vacuum manifold.Following adsorption, the resin is washed five times with 200 μl of 96%(v/v) methanol and the product eluted with 200 μl of 1.0 M potassiumformate in 96% (v/v) methanol. The product is then quantified in afluorescence plate reader, using 466 ηm excitation wave-length andmeasuring emission at 536 ηm. Different enzyme preparations have beenused for this and other (earlier) IPC synthase assays, resulting in dataof varying quality (e.g., Ko et al., 1994; Ko et al., 1995; Mandala etal., 1997; Mandala, et al., 1998; Fischl et al., 1999; Zhong et al.,1999; Heidler and Radding, 2000). Recently, changes in the procedureused for preparation of the enzyme drastically improved the performanceof this type of IPC synthase assay and transformed it from an assayprocedure generating poorly reproducible and at best qualitative data,into a reliable, reproducible assay capable of actually generatingkinetic parameters (Aeed et al. 2004).

Still, a remaining weakness associated with the assay system developedby Aeed et al. (2004) is the complex product work-up procedure.Consistently pipetting (exact amounts of) ion-exchange resin into96-well plates is difficult. And the many manipulations required for theproduct work-up make the assay labor-intensive. Hence, generatingconsistent, reproducible data (with this assay system) is challenging.It depends to a large extent on the skill of the individual carrying outthe work, and the assay is virtually impossible to robotize for use inhigh-throughput screening efforts.

An assay of this invention and described herein circumvents the currentproblems with the IPC assay product work-up. The assay of this inventionincludes reacting phosphatidylinositol with C₆-NBD-ceramide in thepresence of a detergent treated enzyme preparation. The resulting assayproduct does not have to be isolated for quantification. Instead, at thecompletion of the enzymatic reaction, a poly-arginine peptide (or otherphosphate-binding compound) conjugated to a fluorophor (FIG. 3), isadded to the reaction mixture. The poly-arginine peptide (or otherphosphate-binding compound) binds to the phosphate group in the reactionproduct (C₆-NBD-inositol phosphorylceramide). This brings the fluorophor(linked to the peptide or other phosphate-binding compound) in closeproximity to the fluorescent NBD tag on the ceramide portion of thereaction product. One fluorophor that can be used is the7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin[D-346]-hexa-arginine conjugate (shown in FIG. 3) on the peptide (orcompound). The fluorophor is chosen such that it has an emissionspectrum that to a very large extent overlaps the excitation spectrum ofNBD. Consequently, the two fluorescent moieties forms a “fluorescentenergy transfer” (FRET) pair, i.e. the emitted radiation from the higherenergy fluorophor (e.g. D-346) serves as excitation radiation for thelower energy moiety (e.g. NBD) (FIG. 4). And when the reaction mixture(following addition of the fluorophor-conjugated polyarginine peptide)is illuminated at the excitation wavelength of the polyargininepeptide-conjugated fluorophor, a fluorescence signal, proportional tothe amount of reaction product in the mixture, will be emitted at theemission wave-length of NBD. In practical terms, this means that theamount of product formed in the assay can be quantified directly in the96-well (or 384-well) assay plate, without separation from the reactionmixture or any other work-up, using a 96-well plate fluorescence reader.Hence, an assay comprising this product quantification method is readilyadaptable to robotized high-throughput screening efforts.

An additional advantage of the FRET-based assay is that the FRET signalused for quantification of the reaction product is quite specific. Itwill be generated only if the two fluorescent moieties are brought inclose proximity of each other, i.e., the fluorophor-conjugated peptide(or other fluorophor-conjugated phosphate-binding compound) by itselfdoes not generate this signal, nor does it generate a signal if bound toany other molecule than the NBD-tagged reaction product. Hence, the datascatter caused by variations in the sample work-up in existing assays,is eliminated.

Different lengths and composition (number of arginine residues) can beused for the polyarginine peptide. Moreover, other (positively) chargedamino acids, such as for instance lysine or histidine, may also be usedin the peptide, either exclusively or in combination with differentcharged or uncharged amino acids, including arginine. The amino acidscan be any amino acids, including naturally occurring and non-naturallyoccurring and modifications and derivatives thereof. The length andcomposition of the peptide is chosen such that adhesion to the reactionproduct is maximized and non-specific binding (to other molecules) isminimized. A peptide containing six arginine residues (FIG. 3) isfunctional. In addition, other compounds, with charged functionalitiescapable of binding to phosphate groups may be used instead of a peptide.

Different fluorophors may be attached, at either end, to the peptide orother phosphate-binding molecule, using known methods. These fluorophorsmay have an emission spectrum that partly or completely overlaps theexcitation (or absorption) spectrum of the fluorophor conjugated toceramide. If an NBD-conjugated ceramide, such as C₆-NBD-ceramide, isused, a functioning example of such a compound is D-346 (FIG. 4).Alternatively, the fluorophor conjugated to the phosphate-bindingpeptide (or other phosphate-binding compound) may have an excitationspectrum that partly or completely overlaps the emission spectrum of NBD(or other fluorophor conjugated to ceramide). Some non-limiting examplesof thiol-reactive fluorophors, i.e. fluorophors that are easilyconjugated to a synthetic peptide are listed in Table 1.

TABLE 1 Examples of Commercially Available Thiol-Reactive FluorescentCompounds Abs Max Em Max Compound (ηm) (ηm) Alexa Fluor 350 346 442Anilinonaphtalene 326 462 Dansyl 328 563 Dapoxyl 374 572 Dibromobimane394 490 Diethylaminocoumarin 384 470 Dimethylaminocoumarin 394 465Dimethylaminonaphtalene 391 500 Monobromobimane 394 490 Monochlorobimane394 490 Naphtalene 336 490 Pyrene 339 384 Stilbene 329 408

Provided the fluorescence characteristics of the fluorophor conjugatedto ceramide are matched to the fluorophor conjugated to the peptide (orother phosphate-binding compound), such that a FRET pair is formed,ceramide-fluorophor conjugates other than C₆-NBD-ceramide can also beused in the assay. Non-limiting examples of commercially availableceramide-fluorophor conjugates are listed in Table 2. However, it isimportant to realize that chemistry approaches suitable for conjugationof a range of different lipid-compatible fluorophors, to ceramide, arereadily available. Hence, a number of fluorophors, in addition to thetwo used for the compounds listed in Table 2, can be used and the onlysignificant requirement for the fluorophor conjugated to ceramide, isthat it is capable of forming an efficient FRET pair with thefluorophore used on the assay detection and quantification peptide orother compound. A few non-limiting examples of fluorophors that likelywould work with a D-346-containing peptide (or other detection compound)when conjugated to ceramide are Alexa Fluor, Fluorescein, LuciferYellow, Oregon Green and PyMPO(1-(3-(succinimidyloxycarbonyebenzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumbromide).

TABLE 2 Examples of Commercially Available Fluorophor-ConjugatedCeramides Abs Max Em Max Compound (ηm) (ηm) C₅-NBD-ceramide 478 541C₆-NBD-ceramide 478 541 C₁₂-NBD-ceramide 478 541 BODIPY FL C₅-ceramide505 513 BODIPY FL C₆-ceramide 505 513 BODIPY TR C₆-ceramide 544 570

The assay comprises incubating a sample with a fluorescent labeled IPCsynthase substrate and adding a phosphate binding compound conjugated toa fluorophor, wherein the phosphate binding compound conjugated to afluorophor forms a FRET pair with the fluorescent labeled IPC synthasesubstrate. The sample can be any material that contains IPC synthase.

The assay can be used to identify an IPC synthase modulator. Themodulator can inhibit or activate IPC synthase. A test compound can beadded to the assay to determine whether it modulates IPC synthaseactivity. Known modulators of activity can be used as controls or usedfor comparison of test compounds. Known inhibitors include, withoutlimitation, Aureobasidin A (Nagiec, et. al. 1997), galbonolide A orrustmicin (Mandala, et al. 1998), and khafrefugen (Mandala, et al.1997).

The present invention also provides kits for assaying IPC synthaseactivity and for testing compounds for modulating IPC synthase activity.The kits can include reagents for performing the assay including one ormore of: (1) a fluorescent labeled IPC synthase substrate, (2) aphosphate binding compound conjugated to a fluorophor, wherein thephosphate binding compound conjugated to a fluorophor forms a (FRET)pair with the fluorescent labeled IPC synthase substrate, (3)phosphatidyl inositol, (4) a sample that contains IPC synthase, (5) aknown modulator of IPC synthase, (6) buffers and other reagents,including, for example, phosphate buffer, and CHAPS and (7) vessels inwhich to perform the assay.

Example Assay Procedure

The conditions described by Aeed et al. (2004) are used to isolate 10 μg(protein) of CHAPS-washed membranes which is then pre-incubated with 4ηmoles of phosphatidylinositol (PI) in 28 μl 71.4 mM potassium phosphatebuffer, pH 7.0 for 30 min in a 96-well plate. The enzymatic reaction issubsequently started by addition of 12 μl of 0.1 mg/ml C₆-NBD-ceramide,in ethanol, or 2 mM CHAPS. Final assay volume is 40 μl and final reagentconcentrations are 50 mM potassium phosphate, pH 7.0, 0.25 mg membraneprotein/ml, 5 μM C₆-NBD-ceramide, 100 μM PI, 0.3% (v/v) ethanol and 0.6mM CHAPS. Following incubation at room temperature for 5-30 min, thereaction is stopped by adding 150 μl 96% (v/v) methanol. 150 μl 100 μMD-346-hexa-arginine in 50 mM potassium phosphate, pH 7.0, is added, andthe plate is incubated at room temperature for 5 minutes. Fluorescenceis subsequently measured at 600 ηm, using an excitation wavelength of384 ηm.

The assay may be scaled up or down, using different volumesappropriately. The assay may also be carried out in any suitable vessel,including plates containing multiple wells, including two up to 384 ormore well plates, microscope slides, or other vessels.

Other Embodiments

The foregoing example and description are not meant to limit theinvention. While the invention has been described in terms of differentspecific embodiments and examples, those skilled in the art willrecognize that various changes and modifications can be made throughroutine experimentation without departing from the spirit and scope ofthe invention. Accordingly, the invention should be understood as notbeing limited by the foregoing detailed description, but as beingdefined by the appended claims and their equivalents.

REFERENCES

The following patent documents and journal articles are herebyincorporated by reference in their entirety.

Patent Documents:

-   WO 2004/050685 PCT/US2003/038595

Journal Articles:

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1. A method for measuring inositol phosphorylceramide (IPC) synthaseactivity comprising: a) providing a sample containing inositolphosphorylceramide synthase; b) providing a fluorescent labeled IPCsynthase substrate; c) providing a phosphate binding compound conjugatedto a fluorophor, wherein the phosphate binding compound conjugated to afluorophor forms a fluorescence resonance energy transfer (FRET) pairwith the fluorescent labeled IPC synthase substrate; and d) measuringfluorescence.
 2. The method of claim 1, wherein the fluorescent labeledIPC synthase substrate is a fluorescent labeled ceramide.
 3. The methodof claim 1, wherein the phosphate binding compound comprises a peptidethat includes at least one positively charged amino acid.
 4. The methodof claim 1, wherein the phosphate binding compound comprises a peptidethat includes one or more of arginine, histidine and lysine.
 5. Themethod of claim 3, wherein the phosphate binding compound comprises ahexapeptide.
 6. The method of claim 2, wherein the fluorescent labeledceramide is selected from the group consisting of C5-NBD-ceramide,C6-NBD-ceramide, C12-NBD-ceramide, BODIPY-FL-C5-ceramide,BODIPY-FL-C6-ceramide and BODIPY-TR-C6-ceramide.
 7. The method of claim2 wherein the fluorescent labeled ceramide is C6-NBD-ceramide.
 8. Themethod of claim 1 wherein the fluorophor conjugated to the phosphatebinding compound is selected from the group consisting of Alexa Fluor350, anilinonapthalene, dansyl, dapoxyl, dibromobimane,diethylaminocoumarin, dimethylaminocoumarin, dimethylaminonapthalene,monobromobimane, monochlorobimane, napthanlene, pyrene, stilbene andD-346.
 9. The method of claim 1 wherein the fluorophor conjugated to thephosphate binding compound is D-346.
 10. The method of claim 1 whereinthe fluorescent labeled substrate is C6-NBD-ceramide and the fluorophorconjugated to the phosphate binding compound is D-346.
 11. The method ofclaim 1 wherein the sample containing inositol phosphorylceramidesynthase is derived from fungal cells.
 12. A method for identifyinginositol phosphorylceramide synthase inhibitors comprising a) providinga sample containing inositol phosphorylceramide synthase; b) providing atest compound to the sample; c) providing a fluorescent labeled IPCsynthase substrate; d) providing a phosphate binding compound conjugatedto a fluorophor, wherein the phosphate binding compound conjugated to afluororphor forms a fluorescence resonance energy transfer (FRET) pairwith the fluorescent labeled IPC synthase substrate; and e) measuringfluorescence.
 13. A kit comprising a) a fluorescent labeled IPC synthasesubstrate and b) a phosphate binding compound conjugated to afluorophor, wherein the phosphate binding compound conjugated to afluororphor forms a fluorescence resonance energy transfer (FRET) pairwith the fluorescent labeled IPC synthase substrate.
 14. The kit ofclaim 13 further comprising a sample containing inositolphosphorylceramide synthase.
 15. The kit of claim 13 further comprisingphosphatidyl inositol.
 16. The kit of claim 14 further comprising aknown modulator of IPC synthase.